Injectable fillers: current status, physicochemical properties, function mechanism, and perspectives

With the increasing understanding of the aging process and growing desire for minimally invasive treatments, injectable fillers have great potential for correcting and rejuvenating facial wrinkles/folds and contouring the face. However, considering the increasing availability of multiple soft tissue fillers, it is important to understand their inherent biophysical features and specific mechanism. Thus, in this review, we aim to provide an update on the current injectable filler products and analyze and compare their critical physicochemical properties and function mechanisms for volume-filling. Additionally, future trends and development processes for injectable fillers are also proposed.


Introduction
Aging is a complex phenomenon that is inuenced by many factors, including genetics, age, diseases, environment, and living habits. Generally, the signs of facial aging include the loss of subcutaneous volume, decrease in elasticity and moisture level in the skin, and an increase in folds and wrinkles. Histologically, this is related to epidermal thinning, dermal atrophy, loss of the elastic tissues within the dermis, and actinic alterations in dermal collagen loss. Consequently, many cosmetic strategies, such as medicine, radiofrequency and implantable biomaterials, have been employed to compensate and correct the signs of aging and restore facial rejuvenation. Among them, injectable llers have received increasing interest because of their unique characteristics such as easy and minimally invasive procedures.
Injectable llers have a long history, beginning with autologous fat, liquid paraffin, and silicone oil. However, some of them have been prohibited due to their numerous complications such as hypersensitivity responses and inammatory reactions leading to ulcerations, stulas, and skin necrosis. The concept of an ideal ller has been debated for many years, including effective, nonimmunogenic, nontoxic, noncarcinogenic, nonmigratory, easily applied, non-palpable, painless, and long lasting llers. To date, an increasing number of injectable llers is emerging and being applied in the commercial market. All injectable llers are categorized as class III medical devices by regulatory authorities to supervise the medical aesthetics.
Injectable llers are materials that are injected in or beneath the skin layers to restore the lost volume, smooth lines, soen creases, and enhance facial contours. In general, injectable llers replenish the lost volume in two ways, i.e., physical lling and stimulating the synthesis of new collagen. The former shows a greater lling effect aer injection but disappears gradually. The classical llers include hyaluronic acid (HA) and collagen, where the latter stimulates collagen formation by creating a space and structure for the entry of broblasts and vascular cells, making them more effective in the later phases of injection. The typical llers are composed of polymer microspheres suspended in solution, such as polylactic acid (PLA), polycaprolactone (PCL), hydroxyapatite (CaHA), and polymethyl methacrylate (PMMA).
Considering characteristics of materials and their mechanisms of action, in this review, we classify polymeric injectable llers into two categories, i.e., physical llers and biostimulatory llers. Furthermore, the physical llers are subgrouped into HA-based llers and collagen-based llers. In addition, we thoroughly analyse the function mechanisms, vital properties, and perspectives of injectable llers, providing fundamental and constructive suggestions for facial rejuvenation.

Physical fillers
Hyaluronic acid (HA)-based llers HA, the main component of the extracellular matrix (ECM), is a linear polymer belonging to the class of glycosaminoglycan heteropolysaccharides (GAGs). The structure of HA is composed of repeated disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating b-1,3 and b-1,4 glycosidic bonds. 1 Endogenously, a human weighing 70 kg has around 15 g of HA, with about half of that contained in the skin. 2 Accordingly, most injectable ller products are HA based, and presently the most widely utilized. Aer supercial injection, HA can enhance skin tone and elasticity, while supplements of vitamins, amino acids, and peptides can provide nutritional ingredients to support its positive effects. However, noncrosslinked HA has a limited half-life in the body of around 1-2 days. It degrades rapidly via the scission of its glycosidic bonds caused by endogenous hyaluronidase and reactive oxygen species. 3 Moreover, its elasticity is insufficient to li tissues, limiting its utility as a ller.
Thus, to overcome these aws, the structure of HA is modied to fabricate gels with a prolonged residence duration and enhanced viscoelastic properties. Faivre et al. summarized the multiple chemical routes that can be employed for the preparation of HA hydrogels, as shown in Fig. 1. 4 Among them, 1,4butanediol diglycidyl ether (BDDE) and divinyl sulfone (DVS) are the most common crosslinking agents applied in chemical modications and crosslinking, while the former is more predominate due to its lower toxicity and better reactivity. 5 The epoxide groups at the two ends of the BDDE molecule preferentially form an ether bond with the most available primary alcohol in the backbone, making BDDE-crosslinked HA llers durable for up to 1 year. However, considering the mutagenic and carcinogenic potential of BDDE, regulated medical devices require the absence of residual BDDE, and if not, a content considerably below its toxicity threshold of 2 ppm in the nal gel. 6 Presently, multiple commercial HA-based injectable ller products are available, where the main differences in their parameters are their source, concentration, particle size, molecule weight, crosslinking agent, technology for the crosslinking of HA, and the existence of non-crosslinked HA phases.
Tables 1 and 2 present the representative HA ller products approved in the United States and China. [7][8][9][10][11][12] All experimental parameters including HA concentration, molecule weight, particle size, and cross-linking degree impact the physicochemical properties of the product, determining its usage and therapeutic consequences. The crucial physicochemical properties of some products including rheology, cohesivity, extrusion force and swelling factor are summarized in Table 3.
The rheology property of a product is correlated with the ow and deformation of its materials in response to stress. The elastic modulus (G ′ ), which is also known as the storage modulus, measures the hardness of a material and its capacity to resist deformation. G ′ represents numerous factors that affect gel strength, and thus has become a major parameter to differentiate products. The viscous modulus (G ′′ ), which is also called the loss modulus, refers to the energy dissipated by friction throughout a stress cycle and represents the failure of a gel to entirely recover its shape aer the shear force is removed. In terms of rheologic tailoring, rmer gels with a greater G ′ are more resistant to deformation but may feel lumpier aer implant and produce more adverse effects such as pain, inammation, and edema. Thus, products with a high G ′ are employed for contouring and sculpting in deeper areas, such as the chin. Conversely, llers with a lower G ′ are soer and provide a more natural feel upon implantation, making them more suitable for the treatment of so tissue and super-cial zones such as the lip, glabella, and periorbital area. Intermediate G ′ llers can be utilized for dynamic wrinkle correction, as well as supporting and contouring in regions of facial animation, such as the midface. 13,16 Furthermore, the addition of lidocaine can signicantly minimize the discomfort to patients during injection without substantially affecting the rheologic properties of the llers.
Cohesivity characterizes the internal adhesion forces between the cross-linked HA particles that remain intact and not dissociated under an external force. Based on photographs, the ve principal patterns of gel behavior are listed in Fig. 2. 17 Usually, the cohesivity of a hydrogel is calculated based on the average drop in its weight in a syringe under stress. 8 It has been reported that products with a higher cohesivity have higher integration and li capacity. Low-cohesivity llers are generally recommended for modest rhytid correction because they are easier to shape and disperse uniformly in the skin. Fillers with high cohesivity are more suitable for re-volumizing larger areas of loss. 18 A dermal ller must be cohesive enough to withstand compression forces aer injection, avoiding its migration. 19 The extrusion force is the force exerted by HA gels injected through a syringe needle with a specic size, which is determined by G ′ , particle size and distribution range. Currently, there are no international standards or methods to govern the injection of HA gels; however, various syringeability and injectability tests can be found in the literature. 15,20,21 Gels with larger particles or G ′ will be more difficult to inject through a small-bore needle. In the extrusion process, a limited range of particle size distribution can reduce interruptions and uctuations in force. To facilitate particle extrusion, stiff gels with  a greater G ′ must be designed as smaller particles with a narrower particle size range or incorporation of a tiny quantity of non-crosslinked HA chains to lubricate and uidize the gel. Soer gels with a low G ′ can have a wider particle size range and can be easily distorted as they pass through the needle. 22 The swelling factor, which is also known as the gel uid absorption, characterizes the capacity of a gel to expand as it binds water, while remaining a single phase in vitro. The swelling factor was evaluated by thoroughly mixing 0.5 g of gel with 6 to 8 mL of saline, which then increased to 10 mL. 14 When the swelling factor is close to equilibrium, a gel will not swell signicantly aer injection; otherwise, it will rapidly absorb water from the surrounding tissue uid. The swelling factor varies between products and is affected by the concentration of HA and the physical limits imposed by crosslinking, where G ′ increases and the swelling factor decreases as the degree of crosslinking increases.
Crosslinked HA llers are thought to volumize so tissues due to their water-binding and space-lling characteristics. Many studies have proven that HA injectable llers provide structural support for the extracellular matrix (ECM) in the dermis, consequently stimulating broblast activation and collagen synthesis. Quan et al. injected crosslinked HA llers into aged skin and discovered that the broblasts around the ller had elongated morphologies, indicating the enhanced mechanical forces and structural support within the dermal ECM (Fig. 3). Importantly, broblast elongation is associated with the upregulation of the TGF-b signaling pathway and its downstream targets CTGF/CCN2 and type I procollagen. 23 Another study suggested that HA promotes MAPK/ERK phosphorylation and TGF-b1-dependent broblast proliferation by facilitating interactions between CD44 and EGFR. 24 Thus, the effects of hydration, direct volume lling, and new collagen production appear to represent the results of so tissue augmentation using crosslinked HA llers.
Crosslinked HA ller may elicit allergic symptoms such as temporary erythema, edema, itching, and moderate swelling. Furthermore, due to the anti-coagulant activity of HA and the Tyndall effect, it may occasionally result in the formation of supercial bruises, pale nodules, hypertrophic scars, and tiny discolored regions. Serious injuries have also been documented, including foreign-body granulomas and vascular occlusion.
The most prevalent possible consequences are immunemediated adverse events and inadvertent injection, which tend to resolve spontaneously within a few hours or, at most, a few days. In this case, a skin allergy test performed before treatment can largely prevent foreign-body granulomas. 25 Rapid enzymatic degradation with the appropriate number of hyaluronidases can alleviate vascular occlusions. 26 Hylenex, an FDAapproved human recombinant formulation of hyaluronidases, promotes HA elimination through the kidneys with its effects lasting around 48 hours. 27 Furthermore, these risks can be minimized with a masterful understanding of the facial vascular anatomy. It may also be avoided by the expertise of cosmetic treatment professionals. The crosslinking agent of Hylaform, Hylaform PLUS, Prevelle silk (Hylaform with lidocaine) and Captique is DVS, and the crosslinking agent of Elevess is EDC, and the others is BDDE. The origin of Hylaform, Hylaform PLUS, and Prevelle Silk is rooster comb and the others are non-animal stabilized HA. Mono-phase means the nal product only has the crosslinked HA hydrogel and biphase means the nal product is a mixture of crosslinked HA hydrogel and non-crosslinked HA solution. The origin of these products is non-animal stabilized HA. The crosslinking agent of Hyalomatrix, Matrill and Refairywave is DVS, and the others is BDDE. Mono-phase means the nal product only has the crosslinked HA hydrogel and bi-phase means the nal product is a mixture of crosslinked HA hydrogel and non-crosslinked HA solution.

Collagen llers
Collagen is the main component of the ECM in the human body, providing physical support, great tensile strength, and resilience to tissues and organs. Besides, collagen interacts with a number of macromolecules, including integrins, decorin, bronectin, heparin, and matrix metalloproteases (MMPs) to regulate critical functions during tissue regeneration. Aging is accompanied by the loss, disorganization and fragmentation of collagen. The fragmentation of collagen bers impairs its interaction with broblasts, changing the cell morphology, and thereby reducing the mechanical forces. 28,29 Thus, collagen llers can be utilized to replenish collagen, correct the loss of volume, and maintain the health of the ECM environment. To date, several types of collagen products have been approved to act as so tissue llers. Table 4 reviews the currently approved collagen products.
Collagen ller was initially applied in clinic in 1951, while Zyderm I was the rst FDA-approved collagen ller product in 1981. Following that, Zyderm II and Zyplast emerged successively. 30,31 However, because the collagen in these products is sourced from bovines, intradermal skin allergy testing is necessary before the injection operation. Aer two skin tests, the allergy risk can be lowered from 3% to 0.5%. 32 Subsequently, aiming to decrease the potential immunogenicity, human-derived collagen products were developed, which are known as CosmoDerm I, CosmoDerm II and CosmoPlast. Their specic parameters are similar to that of their    counterparts Zyderm I, Zyderm II and Zyplast. Allergy testing is not necessary when injecting these implants because immunogenicity studies have demonstrated a signicant decrease in potential hypersensitivity reactions (less than 1.3%). 33 Dermologen, Cymetra and Fascian are produced from collagen bers and extracellular matrix derived from human cadaveric tissue. The procedures used to acquire these tissues include cell rupture and removal, prion inactivation, two viral inactivation phases, and lastly sterilization. 34,35 Isologen is a collagen product that is created as an autologous implant derived from the skin of the patients. A 3 mm punch biopsy is normally collected from the area behind their ear and delivered to isologen for culture. Aer weeks of cultivation, 1 to 1.5 cm 3 of broblasts and extracellular matrix components are transported back to the physician for injection. [36][37][38] Isologen has no risk of infectious agent transmission from an animal or human donor, and it does not cause hypersensitivity to foreign proteins. However, it requires several procedures and is very expensive.
In addition to bovine collagen, porcine-derived collagen was employed as a ller product. Fibrel, a lyophilized version of gelatin powder, was authorized by the FDA in 1989 for the treatment of depressed cutaneous scars and for facial lines and wrinkles in 1991. Evolence, which is crosslinked by D-ribose, was CE marked in 2004 and approved by the FDA in 2008 to correct moderate to severe facial wrinkles and folds.
The most signicant disadvantage of collagen products in comparison to HA injectable llers is their immunogenicity. Although telopeptide removal and crosslinking methods can be used to enhance the immunogenicity of collagen, the risk of viral infection remains a challenge because the tolerance of collagen products to terminal sterilization is limited. Accordingly, recombinant human collagen-based products have become the focus of research. Recombinant human collagen (rhCollagen), which is identical in structure and functionality to human collagen, was successfully produced by expressing a particular gene segment transcribed into the host. 39 Unlike tissue extract protein, rhCollagen is not immunogenic and not allergic, and it has an intact triple helix structure that demonstrates superior biological function. Ma et al. synthesized a potential hydrogel based on human-like collagen and chitosan, which can be employed as a dermal ller with a less intense inammatory response in the presence of dialdehyde starch. 40 Seror et al. created photocurable rhCollagen by chemically modifying the protein to facilitate crosslinking under illumination, which was used as a dermal ller and a bioink for 3Dprinted breast implants. 41 Karisma Face RhCollagen is a newly introduced injectable so ller that is comprised of R polypeptide a1 chains of type I collagen, high molecular weight HA and carboxymethyl cellulose (CMC). It restores the rmness and structure of the skin gradually, visibly, and permanently.

Bio-stimulatory fillers
Protein absorption, cell recruitment, and brotic encapsulation are the three sequential stages in the foreign body response to foreign material. Once an implant is injected, microspheres evoke a subclinical foreign body inammatory response, culminating in the encapsulation of the microparticles, followed by broplasia and collagen type I deposition in the extracellular matrix 42 (Fig. 4). Based on this, some biomaterials such as PLLA, PCL, PMMA, and CaHA have been employed as collagen stimulators to augment so tissue volume.

Poly(L-lactic acid) (PLLA)
Poly-L-lactic acid is a biodegradable, biocompatible and synthetic polymer invented by a French chemist in 1954. It has been employed as a resorbable suture material and plates and screws in orthopedic, neurologic, and craniofacial surgeries. Unlike other llers that provide instant correction, the volume of the injected regions increases aer injection due to the mechanical distention from the suspension of the microspheres, followed by new collagen production via broblast activation by the PLLA microspheres. Specically, macrophages and foreign body giant cells detect PLLA as a foreign body and recruit and stimulate broblasts via TGFb1 to proliferate and differentiate into myobroblasts. (Myo-)broblasts encapsulate PLLA particles with collagen type III and deposit brotic collagen type I around the capsule. 43 Importantly, the foreign body response is biocompatible, where PLLA microspheres are degraded slowly to carbon dioxide and water over a period of weeks to months. 44 PLLA microspheres were destroyed completely over the course of 9 months, with no leover PLLA or scarring brosis observed. 45 Sustained volumetric expansion and correction with PLLA have been reported for up to 2 years, although the majority of PLLA would have been entirely metabolized by that time. 46 Sculptra is composed of lyophilized PLLA particles, which was authorized by the FDA for the recovery and correction of adipose atrophy in HIV patients in 2004. Sculptra aesthetic was approved in 2009 for grid injection into the deep dermis to treat moderate to severe nasolabial folds and other wrinkles/folds. Each Sculptra syringe has 150 mg PLLA microspheres and 217.5 mg sodium carboxy-methylcellulose and mannitol. Before injection, PLLA powders must be reconstituted 24 to 72 h to fully hydrate and form a very viscous hydrogel. The vial should be vigorously shaken just before injection to ensure homogenous mixing. 47 Löviselle, which was approved by NMPA in 2021, consisted of mixed freeze-dried PLLA microspheres powder, mannitol, and sodium carboxymethyl cellulose. These freezedried powders need to be dissolved with 0.9% sodium chloride before use.
PDLLA (poly-D,L-lactic acid) is also a biocompatible, biodegradable, bio-stimulatory and long-lasting material that can be used as a new subdermal stimulatory ller. AestheFill is a ller composed of 30 to 70 mm PDLLA microspheres suspended in sodium carboxymethylcellulose. Lin et al. injected AestheFill into SD rats and identied extracellular type I collagen in areas between and on the outside surfaces of the microspheres aer 4 weeks and inside the individual microspheres by 20 weeks (Fig. 5). 48 The short-term adverse effects following the injection of PLLA and PDLA microspheres include moderate transitory localized erythema, ecchymosis, and edema, as predicted. Nonvisible, palpable subcutaneous nodules, which typically disappear spontaneously, are some of the long-term adverse effects, together with chronic granulomatous responses (0.2-1.2% incidence). 49

Polycaprolactone (PCL)
Poly-caprolactone (PCL) is a biocompatible, biodegradable, and bioresorbable polymer widely used in surgical sutures, articial blood vessels/skin, bone and so tissue llers, and tissue scaffolds. Similar to PLLA, the degradation end products of PCL are CO 2 and H 2 O, which can be totally eliminated from the body. 50 Ellanse, which consists of 30% PCL microspheres and 70% carboxymethylcellulose (CMC) gel carrier, presents different functions. The CMC gel is responsible for the immediate impact due to the injected volume lling capacity and very hygroscopic feature of CMC, but it is resorbed within 2-3 months. The PCL microspheres have a long-lasting impact due to the synthesis of collagen and scaffold formation. Based on the chain length (molecular weight) of the initial PCL chains within the microspheres, Ellanse is categorized into four models with durations ranging from 1 year to 4 years.
Kim et al. conrmed the dual-effect of EllanséTM-M, i.e., direct and "delayed" volumizing effect. According to human biopsies at 13 months, new collagen formed around the PCL particles through the activation of neocollagenesis (Fig. 6). 51 Kim further evidenced the collagen production and skin texture improvement in the human temple aer treatment with Ellansé-M. According to biopsies, new collagen bers, elastic bers, and neovascularization with new capillaries were observed at 1 year and 4 year post-treatment 52 (Fig. 7).
To further identify the type and content of collagen synthesis, Oh et al. used Masson's trichrome (MT) and Sirius   red (SR) staining. MT staining revealed that the number of nuclei increased in both groups as the content of collagen increased following broblast stimulation by the microspheres. SR demonstrated that type III collagen (thin green and yellow color) was freshly generated aer 8 weeks of injection, and thicker and mature red and reddish-yellow type I collagen bers were created over time (Fig. 8). 53 Globally, Ellanse has been shown to have no serious adverse effects, granuloma or vascular complications, with only a few initial injection-related responses, mostly edema or ecchymosis, which are generally minor and resolve naturally without intervention within a few days.

Calcium hydroxylapatite (CaHA)
Calcium hydroxylapatite (CaHA) is a component of human bone and teeth, and synthetic CaHA has been utilized in medicine as a biodegradable and biocompatible substance for more than 20 years. CaHA is the main ingredient in Radiesse to treat moderate to severe facial wrinkles and folds. Each Radiesse syringe consists of 30% CaHA microspheres suspended in 70% carrier solution including sterile water, glycerin, and carboxymethylcellulose. Radiesse has double effects, including immediate mechanical lling of the carrier solution and long-term neocollagenesis through the CaHA microspheres. Between them, the immediate lling effect will be absorbed with time and replaced by collagen due to the stimulation of the CaHA microspheres.
Aer injection, the deposited CaHA particles can mimic the host environment and support the ingrowth of broblasts and collagen. The lling-up period is up to 2 years due to the slow enzymatic metabolism and phagocytosis of the CaHA microspheres. 54 The CaHA microspheres are eventually totally degraded into calcium and phosphate ions and secreted by the body, following the same metabolic process as the bone debris produced by typical bone fractures. 55 Lorenc et al. evaluated the broblastic response of Radiesse and discovered that broblasts surrounded the CaHA microspheres aer 3 weeks, and 6 months later, the microspheres varied in size when they are broken up by the brous connective tissue reaction and surrounded by brous connective tissue (Fig. 9). 56 Radiesse does not require skin testing because it is immunologically inactive. However, its adverse events are the same as other semi-permanent llers such as PLLA and PCL microspheres. Its short-term side effects include moderate, temporary localized erythema, ecchymosis, and edema. Its long-term adverse effects include nodules and granulomas, particularly when injected into the lip. Broder found that Radiesse dissipates soon aer injecting into the lips and the CaHA particles cluster together, generating noticeable hard white nodules in the lip. 57

Poly(methyl methacrylate) (PMMA)
Polymethylmethacrylate has been used successfully in medical implants such as orthopaedic bone cement and craniectomy plates for more than 65 years. Compared with other  biomaterials for the treatment of wrinkle lines and so tissue loss, which degrade within a few months to years, PMMA is a non-resorbable synthetic chemical with a permanent effect. 58 Artecoll is a permanent ller made up of 20% PMMA microspheres suspended in an 80% bovine collagen solution (3.5% bovine collagen, 2.7% phosphate buffer, 0.9% sodium chloride, 0.3% lidocaine hydrochloride and 92.6% water). 59 ArteFill is another version of Artecoll that was rebranded as Bellall in 2014. 60,61 Aer deep dermal injection of these products, the collagen carrier is degraded by the body and totally replaced by the its own collagen at the same pace, resulting in consistent augmentation. The permanent PMMA microspheres can be considered as "living implants" that provide tissue augmentation through broplasia with a 5 year lasting effect.
ArteFill (Bellall) has also been evaluated for its effectiveness and safety in the correction of atrophic facial acne scars. In a double-blind, randomized, multi-center, control study, 64% of the ArteFill-treated participants and 33% of control (saline) subjects were successful. 62 Lemperle et al. further revealed that all the PMMA microspheres are entirely encapsulated and surrounded by broblasts and collagen bers, with only a few macrophages at 3 months. Strong bands of mature collagen bers with fully intact capillary vasculature could still be seen surrounding the intact PMMA microspheres aer 10 years (Fig. 10). 60 Its short-term side effects include expected mild, temporary localized erythema, ecchymosis, and edema. The predominant long-term adverse event documented in a 5 year safety and satisfaction study evaluating the use of Bellall in the treatment of nasolabial folds is the formation of granulomas, which had an overall incidence rate of 1.7%. 63 Furthermore, PMMA is a key component in Metacrill and NewPlastic. In South America, Metacrill, a so tissue ller composed of PMMA microparticles suspended in a carboxymethylcellulose colloid, has been utilized to treat facial rhytids, acne scars and facial herniatrophy. 64 The microparticles range in size from 1 to 80 mm and have an uneven shape (Fig. 11). 61,65 NewPlastic consists of PMMA particles suspended in sodium hyaluronate (2%), D-1 propanediol (10%), and a pyrogenous solution. 66,67 The microspheres of NewPlastic are 30-70 mm in size and non-spherical, with conjoined particles. Thus, due to the particle size, morphology, and surface characteristics of these two products, there are not as popular as Bellall.
For bio-stimulatory injectable llers, several key parameters such as the size and morphology of the particles, the viscosity of the carrier gel and rheology property inuence their longevity, inammatory, and function.
When the diameter of a microsphere is in the range of 15-20 mm, it is at risk of being phagocytosed by macrophages, resulting in giant cell formation and granulomatous inammation. 60 Microspheres with diameters smaller than 10 mm have also been demonstrated to promote lymphocyte inltration and vascularization. Lemperle et al. reported that the smaller the microspheres (to the threshold of phagocytosis), the larger their combined surface area in a given volume and the greater the total amount of new collagen formation. Microspheres with a mean diameter of 100 mm stimulate only about 56% connective tissue, whereas microspheres with a mean diameter of 40 mm promote around 80% connective tissue ingrowth. 68 However, oversizing of the microspheres may cause a serious inammatory reaction. In addition, research has shown that allogeneic giant cells cluster near particles with uneven surfaces, and therefore the smoothness and uniformity of the individual microspheres minimize the inammatory response. 69 Thus, microspheres with diameters ranging from 20 to 50 mm appear to be excellent for cutaneous injections.   Microspheres in this distribution are large enough to avoid phagocytosis, while remaining tiny enough to be effortlessly delivered through a ne 26G or 30G needle with no need for considerable force. Gold et al. also found that the diameter of PMMA microspheres between 30-50 mm was optimal for maximizing the surface area exposed to autologous collagen. 61 Strikingly different from HA, the key components of these injectable llers are relatively low hydrophilicity. Thus, these microspheres easily deposit when mixed with a solution with low viscosity, such as water and PBS buffer solution. However, a carrier solution with excessive viscosity will increase the injection force for extrusion through a needle. Thus, it appears that the appropriate viscosity of the carrier solution is critical in ensuing the uniform dispersion of microspheres, which enables tissue formation in the interlayer. Besides, the viscosity of the carrier gel, in which the microspheres are uniformly embedded, prevents clumping of the particles during the formation of the host tissue matrix. 67 Table 5 summaries the representative injectable llers approved in the United States.
Similar to HA llers, understanding the rheological characteristics of biomaterial microspheres is critical because they can impact the ller performance. However, the rheological features of regeneration llers are less investigated compared to that of HA llers. Lorenc et al. investigated the li capacity, deformation resistance, tissue integration and physicochemical properties of different HA llers and CaHA using three animal models 56 (Fig. 12). CaHA outperformed the HA llers in terms of G ′ , immediate resistance to deformation, and sustained cohesivity at all time points. However, the larger G ′ associated with CaHA did not always imply a stronger li capacity compared to the HA llers. The dramatic difference in G ′ and viscosity between CaHA and HAs shows that they may be regarded complementary rather than competing. 70

Others
Outline/evolution is a copolymer of diallyldimethylammonium chloride and acrylamide that has been partially crosslinked by N,N ′ -methylenebisacrylamide and PVA hydrogel microspheres (5-40 mm). Because the copolymer and PVA have a positive charge, negatively charged tissue molecules such as hyaluronic acid and other amino acids glucan are drawn to them. Two months later, negatively charged tissue molecules progressively enter the implant and produce so spongy material to improve the face contour.
Aquamid is a biocompatible, non-absorbable hydrogel made by polymerizing acrylamide monomers with N,N ′ -methylenebisacrylamide. Aquamid is composed of 2.5% crosslinked polyacrylamide and 97.5% water, which has been widely used for the treatment of different rhytids, facial contouring and correction, and its efficacy can last for more than 1 year. In 37 cases, the most common adverse effects were erythema, bruising, swelling, itching, and discomfort. A modest color change at the injection site and one incidence of neutropenia were among the unusual adverse events identied. 71 Perspectives Besides the products described above that have been approved by the FDA, CE or NMPA, many modication strategies are being applied to reduce the complication rate and improve the properties of llers, and new diverse materials are being researched to deliver ideal tissue regeneration.
The modication of molecular structure can avoid their shortcomings. Commercial HA-based llers are made using crosslinking agents such as DVS and BDDE, which increase the toxic risk. To avoid the need for harmful chemical crosslinkers, Hong et al. modied an HA derivative with catechol groups that may self-crosslink via self-oxidation. 72 Hong et al. used less toxic vitamin B2 derivatives as photo-initiators to introduce tyramine into HA to impart photo-crosslinking ability. 73 To further extend the duration, amino acids were graed onto HA to signicantly reduce its enzymatic degradation. 74 Schanté et al. demonstrated that amino acid-modied HA derivatives are good materials for biomedical applications, particularly HA-tyrosine, which also exhibited increased resistance to enzymatic digestion in a variety of amino-acid modied HA hydrogels. 75 Because of the super-hydrophobicity of PLLA and PCL, their microspheres are different to mix uniformly in carrier solution, and hence polyethylene glycol (PEG) was introduced to increase the water solubility. 76 Steinman et al. prepared a triblock copolymer of PCL-PEG-PCL with different molecular weights of PEG to increase the water solubility and showed its potential as dermal llers. 77 Cui et al. synthesized hydrogels composed of PLA-PEG-PLA copolymer with good water-solubility, which can be used in tissue engineering. 78 CureWhite, a PLLA-PEG microsphere suspension in crosslinked HA hydrogels approved by NMPA in 2021, is indicated for the correction of moderate to severe nasolabial folds and wrinkles.
Besides the above-mentioned biomaterials, researchers are always exploiting new materials. Lee et al. suggested that autologous platelet-rich plasma (PRP) plays an essential role in increasing collagen expression, matrix remodeling proteins, broblast proliferation and differentiation into myobroblasts. 79,80 Kang et al. identied keratin-brinogen hydrogels as potential ller materials to accelerate tissue regeneration. 81 Choi et al. created injectable and physical hydrogels by mixing levan with Pluronic and CMC, demonstrating the potential of levan as a novel material for dermal llers. 82 Kim et al. fabricated an HA-PN (hyaluronic acid-polynucleotide) complex ller improvement in periorbital skin accidity in ve patients. 89 Rhee et al. developed a long-term ller system comprised of a blend of HA ller and live human mesenchymal cells to maintain the impact tissue augmentation. 90 Huang et al. demonstrated that human adipose stem cells proliferated and further differentiated into adipose tissue in HA hydrogels in vitro, retaining the potential of tissue ller. 91 Zhao et al. reported the development of an injectable hydrogel derived from human acellular adipose tissue that may trigger the generation of human adipose stem cells (HASCs). 92 These ller products may not only physically ll or cause a host response, but may also regenerate normal human so tissue and further delay the natural aging processes.
Currently, anesthetics such as lidocaine are widely used with dermal llers to minimize the discomfort during injection, but more drugs may be added in the future to provide other functions. Filler stimulation at the implant site should be evaluated both positively and negatively. Thus, by modulating and adjusting the stimulation, such as enhancing or inhibiting the growth of bers with different drugs, the ller-drug combination may better match the proper host tissue responses. Fan et al. discovered that combining PLA microspheres and PEG-PCL-PEG micelles with dexamethasone may boost collagen production. 93 Olmo et al. loaded the antibiotics cefuroxime (CFX), tetracycline (TCN), and amoxicillin (AMX) as well as the anti-inammatory drug acetylsalicylic acid (ASA) into a crosslinked HA hydrogel to promote the antibacterial and anti-inammatory response. 94 Combination therapies, in addition to combining llers with drugs and medications enable an appropriate response to the multifactorial process of aging, which involves structural changes in all anatomical layers and dynamic interactions among these tissues. [95][96][97] Multiple aims such as relaxing, volumisation, volume relocation, reshaping, resurfacing, or tightening can be achieved using the complete, three-dimensional and multi-layered strategy, which combines multiple agents and procedures. 98,99 Melo concluded that combination therapies have additive or even synergistic benets, resulting in better and longer-lasting therapeutic outcomes than single agent-or single technique-based protocols, with no clinical evidence of increased incidence or severity of side events 100 (Table 6).

Conclusions
For decades, patients and clinicians have been attracted to the use of injectable llers for so tissue augmentation. Multiple types of injectable llers have been invented, optimized and commercialized, while others have vanished from the market for some specic reasons. In this review, we summarized the majority of currently available injectable llers, highlighted critical indicators, and investigated their function mechanisms. The critical parameters including concentration, rheology, microsphere size, and viscosity are the key differences between these products and result in unique actions. Given the current limits, innovative solutions for optimal tissue augmentation generation must be studied to suit the increasingly diverse demands by patients.

Conflicts of interest
There are no conicts to declare.